Pyrazolo[4,3-d]pyrimidines as Antitumor Agents

ABSTRACT

A compound of Formula II, or optionally a salt or a hydrate of the compound of Formula II is provided:wherein R1 is selected from the group consisting of a hydrogen, an alkyl group having from one to six carbon atoms, an aryl group, and a heteroaryl group; R2 is selected from the group consisting of an alkyl group having from one to six carbon atoms, a hydrogen, a NRaRb group wherein Ra is either a hydrogen or an alkyl group having from one to six carbon atoms and Rb is either a hydrogen or an alkyl group having from one to six carbon atoms, an aryl group, a heteroaryl group, and a halogen; R is an alkyl group having from one to six carbon atoms; and Ar is selected from the group consisting of an alkyl group having from one to six carbon atoms, a substituted aryl, and a substituted heteroaryl.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional patent application is a divisional patentapplication of and claims the benefit of co-pending U.S. patentapplication Ser. No. 15/929,363, filed on Apr. 28, 2020, which is adivisional patent application of and claims the benefit of U.S. patentapplication Ser. No. 16/294,372, filed on Mar. 6, 2019, now U.S. Pat.No. 10,689,386, issued on Jun. 23, 2020, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 62/639,654, filed on Mar.7, 2018 (now expired), and to U.S. Provisional Patent Application Ser.No. 62/719,259, filed on Aug. 17, 2018 (now expired). The entirecontents of U.S. patent application Ser. Nos. 15/929,363 and 16/294,372,and U.S. Provisional Patent Application Ser. Nos. 62/639,654 and62/719,259 are incorporated by reference into this non-provisionalpatent application as if fully written herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersRO1CA142868 and RO1A1098458 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention provides pyrazolo[4,3-d]pyrimidine compounds andpharmaceutical compositions and methods of use and manufacture of thesame.

2. Description of the Background Art

Microtubule dynamics is a pivotal factor for controlling cellproliferation. Therefore, suppressing cell proliferation by microtubuletargeting agents is an outstanding way of controlling cell division.¹ Ineukaryotic cells, tubulin heterodimers polymerize to form microtubules.Microtubule formation is a dynamic process which is tightly controlledby cell. Manipulation of the equilibrium between tubulin and microtubuleoffers an array of opportunities to control cell division. Discovery ofnew binding sites in tubulin is an additional impetus for designingmicrotubule targeting agents.

Microtubule targeting agents are mainly classified as two groups,microtubule-stabilizing agents (MSA) and microtubule-destabilizingagents (MDA).² Taxanes belong to the first group which bind to theinterior of the microtubule. They are useful against breast, lung,ovarian and prostate carcinomas. Laulimalide and peloruside A alsobelong to the first group which bind to laulimalide binding site, whichis a unique non-taxane site in ß-tubulin.³ Vinca alkaloids such asvincristine, vinblastine consist of the second group which aremicrotubule destabilizers. These are β-tubulin binding agents used inleukemias, lymphomas and other cancers. Rhizoxin and maytansine bind tothe maytansine binding site in ß-tubulin.⁴ Colchicines comprise of adiverse collection of molecules which bind at the β-tubulin at itsinterface with α-tubulin. These are also microtubule destabilizingagents (MDA). Combretastatin A-4 (CA4) and its phosphorylated analogcombretastatin A-4 phosphate (CA4-P) which bind to the colchicine siteon tubulin are currently in clinical trials. There are no approvedcolchicine site binding agents. This demonstrates the importance ofdeveloping colchicine site agents as antitumor agents.⁵ FIG. 1 shows thechemical structures of known agents.

Mutation in the p53 gene occurs in half of all tumors and microtubuletargeting agents are most effective in treating p53 mutant cells.⁶Multidrug resistance (MDR) is a major limitation in cancer chemotherapy,and MDR tumors are particularly resistant to tubulin-binding agents.⁷Overexpression of P-glycoprotein (Pgp) has also been reported in anumber of tumor types. ⁸ Attempts to reverse drug resistance bycombining antimitotic agents with inhibitors of drug efflux proteinsproduced disappointing results. ³ Expression of ß-III tubulin is anotherclinical mechanism of resistance to tubulin binding agents in multipletumor types including non-small cell lung, ⁹ breast ¹⁰ and ovariancancer. ¹¹Stengel et al.¹² showed that colchicine site binding agentsare the most effective agents against ß-III tubulin resistance whichfurther demonstrates the importance of developing this class of agents.

SUMMARY OF THE INVENTION

The present invention provides compounds of Formula I, andpharmaceutically acceptable salts and hydrates thereof.

wherein X is selected from the group consisting of:

wherein R is an alkyl group having from one to ten carbon atoms, andwherein R₂ is a halogen atom. In a preferred embodiment of thisinvention, the compound of Formula I includes wherein R is a methylgroup. In a more preferred embodiment of this invention R₂ is a halogenatom that is a chlorine. In a most preferred embodiment of thisinvention, the compound of Formula I includes wherein R is a methylgroup and R₂ is a halogen atom that is a chlorine.

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a compound of Formula I, and pharmaceuticallyacceptable salts and hydrates of the compound of Formula I:

wherein X is selected from the group consisting of:

wherein R is an alkyl group having from one to ten carbon atoms, andwherein R₂ is a halogen atom. In a preferred embodiment of thisinvention, the pharmaceutical composition comprises a compound ofFormula I, wherein R is a methyl group. In a more preferred embodimentof this invention, the pharmaceutical composition comprises a compoundof Formula I, wherein R is an alkyl group having from one to ten carbonatoms and wherein R₂ is said halogen atom that is a chlorine. In a mostpreferred embodiment of this invention, the pharmaceutical compositioncomprises a compound of Formula I, wherein R is a methyl group andwherein R₂ is said halogen atom that is a chlorine.

In another embodiment of this invention, a method of treating a patienthaving cancer comprises administering to said patient a therapeuticallyeffective amount of a compound of Formula I to said patient:

wherein X is selected from the group consisting of:

wherein R is an alkyl group having from one to ten carbon atoms, andwherein R₂ is a halogen atom. Preferably, this method of treating apatient having cancer includes wherein the compound of Formula Iincludes wherein R is a methyl group and R₂ is said halogen. In a morepreferred embodiment of the method of treating a patient having cancerof this invention includes wherein the compound of Formula I includeswherein R is an alkyl group having from one to ten carbon atoms and saidhalogen atom is a chlorine. In a most preferred embodiment of the methodof treating a patient having cancer of this invention includes whereinthe compound of Formula I includes wherein R is a methyl group and saidhalogen atom is a chlorine.

BRIEF SUMMARY OF THE DRAWINGS

FIG. 1 shows the chemical structures of known anti-cancer agents.

FIG. 2 shows the chemical structures of known in the art lead compoundsand shows the chemical structures of the target compounds of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “patient” refers to a member of the animalkingdom, including but not limited to, human beings.

As used herein, the term “therapeutically effective amount” refers tothat amount of a compound required to bring about a desired effect in apatient. The desired effect will vary depending on the illness beingtreated. For example, the desired effect may be reducing tumor size,destroying cancer cells, preventing metastasis, or reducing symptomsassociated with various diseases, including but not limited to cancer.It will be understood by a person of ordinary skill in the art that the“therapeutically effective amount” will vary from patient to patientdepending on such factors, for example, but not limited to, the illnessbeing treated, the severity of the illness being treated, and thepatient's ability to mount an immune response. The determination of a“therapeutically effective amount” for a given patient is within theskill of one practicing in the art. Generally, a “therapeuticallyeffective amount” is determined by the potency in standard ex vivocellular systems, and may be followed by pre-clinical and clinical invivo assessment

As used herein, the term “cancer” includes human cancers, for example,but not limited to, leukemia, non-small cell lung cancer, colon cancer,central nervous system cancer, melanoma, ovarian cancer, breast cancer,renal cancer, prostate cancer, and pancreatic cancer. As used herein,the term “having cancer” means that a patient has been diagnosed withcancer.

The present invention provides compounds of Formula I, andpharmaceutically acceptable salts and hydrates of the compounds ofFormula I:

wherein X is one selected from the group consisting of:

wherein R is an alkyl group having from one to ten carbon atoms, andwherein R₂ is a halogen atom. In a preferred embodiment of thisinvention, the compound of Formula I includes wherein R is a methylgroup and wherein R₂ is a halogen atom. In a more preferred embodimentof this invention the compound of Formula I includes wherein R is analkyl group having from one to ten carbon atoms and wherein R₂ is ahalogen atom that is chlorine. In a most preferred embodiment of thisinvention, the compound of Formula I includes wherein R is a methylgroup and R₂ is a chlorine.

In another embodiment of this invention, a pharmaceutical composition isprovided comprising the compound of Formula I, or a salt or a hydrate ofthe compound of Formula I:

wherein X is selected from the group consisting of:

wherein R is an alkyl group having from one to ten carbon atoms, andwherein R₂ is a halogen atom. In a preferred embodiment of thisinvention, the pharmaceutical composition comprises a compound ofFormula I, wherein R is a methyl group and R₂ is a halogen. In a morepreferred embodiment of this invention, the pharmaceutical compositioncomprises a compound of Formula I including wherein R is an alkyl grouphaving from one to ten carbon atoms and R₂ is a halogen atom that is achlorine. In a most preferred embodiment of this invention, thepharmaceutical composition comprises a compound of Formula I includingwherein R is a methyl group and R₂ is said halogen that is a chlorine.

Another embodiment of the pharmaceutical composition comprising thecompound of Formula I, as described herein, includes a pharmaceuticallyacceptable carrier.

Another embodiment of this invention provides a compound of Formula II:

wherein R₁ is selected from the group consisting of a hydrogen, an alkylgroup having from one to six carbon atoms, an aryl group, and aheteroaryl group; R₂ is selected from the group consisting of an alkylgroup having from one to six carbon atoms, a hydrogen, a NR^(a)R^(b)group wherein R^(a) is either a hydrogen or an alkyl group having fromone to six carbon atoms and R^(b) is either a hydrogen or an alkyl grouphaving from one to six carbon atoms, an aryl group, a heteroaryl group,and a halogen; R is an alkyl group having from one to six carbon atoms;and Ar is selected from the group consisting of an alkyl group havingfrom one to six carbon atoms, a substituted aryl, and a substitutedheteroaryl. In a preferred embodiment of this invention, the compound ofFormula II, as described herein, includes wherein R₂ is either a methylgroup, NH₂, or a chlorine. In a more preferable embodiment of thisinvention, the compound of Formula II, as described herein, is oneselected from the group consisting of5-chloro-N-(4-methoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N,1-dimethyl-N-(4-(methylthio)phenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;1-(5-chloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-6-methoxy-1,2,3,4-tetrahydroquinoline;5-chloro-N-(5-methoxynaphthalen-2-yl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N-(4-ethoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N-(4-isopropoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N-(3-fluoro-4-methoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N,1-dimethyl-N-(4-(trifluoromethoxy)phenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;and5-chloro-N-(4-methoxyphenyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine.

As used herein, the term “alkyl” group refers to those alkyl groupshaving from one to ten carbon atoms, and preferably from one to sixcarbon atoms, such as for example methyl, ethyl, propyl, butyl, pentyl,hexyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopropylmethyl orcyclobutylmethyl groups. Alkyl groups sharing one to about six carbonatoms are preferred. These alkyl groups are straight chain, branchedchain or cyclic (alicyclic hydrocarbon) arrangements. The carbon atomsof these straight chain, branched chain or cyclic arranged alkyl groupsmay have one or more substituents for the hydrogens attached to thecarbon atoms.

As used herein, the term “heteroalkyl” refers to alkyl chains from oneto about 3 atoms where one or more of the carbons has been replaced withnitrogen, oxygen or sulfur, Thus “heteroalkyl” groups will include, forexample, —CH₂—CH₂—NH—, —CH₂—S—, —S—CH₂—, —CH₂—O—, —CH₂—CH₂—O—, —O—CH₂—,—NH—CH₂—CH₂—, —NH—CH═CH—, and other various combinations, as will beapparent to one skilled in the art. The above list is not meant to beexhaustive, and many combinations are contemplated as within the scopeof the present invention.

The term “aryl” groups, as used herein, refers to compounds whosemolecules have an aromatic ring structure, such as the six-carbon ringof benzene, or multiple rings which are either fused or unfused, such ascondensed six-carbon rings of other aromatic derivatives. The term“aryl” is also defined to include diaryl, triaryl and polyaryl groups,which would have two, three or more rings, respectively. Thus, suitablearyl groups would include, for example, phenyl, biphenyl, naphthyl,phenanthrene, anthracene groups and aryl oxyaryl groups. This list isnot meant to be exhaustive, and any aryl group, as these terms aredefined above and commonly understood in the art, are within the scopeof the present invention.

The term “heteroaryl” refers to aromatic ring structures having at leastone atom in the ring which is not carbon, such as oxygen, nitrogen orsulfur. “Heteroaryls” as used herein also refers to aromatic ringstructures that are part of larger ring structures, such as two or threemember ring systems, which may be fused or unfused, in which one of therings is as described above. Thus, “heteroaryl” refers to ring systemsin which one or more rings contain a heteroatom and one or more rings donot. It will be understood that this list is not meant to be exhaustive,and that any heteroaryl group, as these terms are defined above andcommonly understood in the art, are within the scope of the presentinvention. The heteroaryl ring systems may be fused ring systems orunfused. Examples of heteroaryl ring systems include, for example butare not limited to, pyridine, quinoline, isoquinoloine, pyrrole,thiophenes, furans, imidazoles, and the like, as well as fused ringstructures having rings of different sizes, such as benzofurans,indoles, purines, and the like.

Also included within the scope of the present invention are alicyclicgroups, as that term is understood in the art, and heterocyclic groups.As used herein, the term “heterocyclic group” refers to non-aromaticcyclic substituents in which one or more members of the ring is notcarbon, and is at least one of an oxygen, sulfur or nitrogen atom, forexample.

The terms “alkylaryl” (or “alkaryl”) or “alkylheteroaryl” as used hereinrefer to groups having an alkyl moiety attached to an aryl or heteroarylring. The alkyl moiety is preferably a straight, branched or cyclicalkyl group having one to about six carbon atoms. This alkyl moiety mayalso contain oxygen, nitrogen or sulfur, and therefore may be an alkoxygroup. The aryl or heteroaryl moiety of the alkylaryl group is asubstituted or unsubstituted aryl or heteroaryl group, as these termsare described above. As used herein, the terms “alkylaryl” or“alkylheteroaryl” will also be used to refer to arylalkyl groups orheteroarylalkyl groups, as those terms are understood in the art, anddenotes attachment of such a substituent at either the alkyl or the arylportion of the group. Thus, for example, a benzyl group would beembraced by the term “alkylaryl”.

Any of the cyclic substituents described above, such as the aryl,heteroaryl, alkylaryl, alkylheteroaryl, alicyclic, or heterocyclicgroups are optionally substituted with one or more substituents aslisted above. In the case of more than one substituent, the substituentsare independently selected. “Alkoxy groups” and “alkyl groups” includestraight or branched chains having up to about ten members.

As used herein, the term “halogen” refers to chlorine, bromine, iodineand fluorine. “Aryl and heteroaryl groups” are as described above. Whena carboxylic acid is a substituent, it will be appreciated that themoiety represents an acid such as benzoic acid.

As used herein, the terms “aroyl” or “heteroaroyl”, are generallydefined in the art as an aromatic or heteroaromatic compound having acarbonyl moiety.

Other embodiments of the present invention provide pharmaceuticallyacceptable salts, and hydrates of the compounds of Formula I and FormulaII. Preferably, the compounds of the present invention represented byFormula I and Formula II may be made into acid salts that are watersoluble. Most preferably, these water soluble salts of Formula I andFormula II may be formulated into an oral dosage forms providing orallyadministered active antitumor agents. In the past, antimitotic agentshave been plagued with water solubility problems, such as for examplebut not limited to Taxol® (Bristol-Myers Squibb Company) andcombrestastatin, and a variety of solubilizing agents have been employedto improve their water solubility. The present salts of the compoundshaving Formula I and Formula II overcome such water solubility problemsand are generally completely water soluble.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients being treated, each unitcontaining a predetermined quantity or effective amount of a compound ofthe present invention to produce the desired effect in association witha pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the particularcompound and the particular effect, or therapeutic response, that isdesired to be achieved.

Compounds containing Formula I or Formula II, or pharmaceuticallyacceptable salts, or hydrates thereof, can be administered to a patient(an animal or human) via various routes including parenterally, orallyor intraperitoneally. Parenteral administration includes the followingroutes that are outside the alimentary canal (digestive tract):intravenous; intramuscular; interstitial, intraarterial; subcutaneous;intraocular; intracranial; intraventricular; intrasynovial;transepithelial, including transdermal, pulmonary via inhalation,ophthalmic, sublingual and buccal; topical, including dermal, ocular,rectal, or nasal inhalation via insufflation or nebulization. Specificmodes of administration shall depend on the indication. The selection ofthe specific route of administration and the dose regimen is to beadjusted or titrated by the clinician according to methods known to theclinician in order to obtain the optimal clinical response. The amountof compound to be administered is that amount which is therapeuticallyeffective. The dosage to be administered to a patient shall depend onthe characteristics of the patient being treated, including for example,but not limited to, the patient's age, weight, health, and types andfrequency of concurrent treatment, if any, of any other chemotherapeuticagent(s), all of which is determined by the clinician as one skilled inthe art.

Compounds containing Formula I or Formula II, or a salt, or a hydratethereof, that are orally administered can be enclosed in hard or softshell gelatin capsules, or compressed into tablets. Compounds also canbe incorporated with an excipient and used in the form of ingestibletablets, buccal tablets, troches, capsules, sachets, lozenges, elixirs,suspensions, syrups, wafers and the like. Compounds containing Formula Ior Formula II can be in the form of a powder or granule, a solution orsuspension in an aqueous liquid or non-aqueous liquid, or in anoil-in-water emulsion.

The tablets, troches, pills, capsules and the like also can contain, forexample, a binder, such as gum tragacanth, acacia, corn starch, gelatingexcipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; a sweetening agent, such as sucrose, lactose orsaccharin; or a flavoring agent. When the dosage unit form is a capsule,it can contain, in addition to the materials described above, a liquidcarrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For example,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the active compound, sucrose as a sweeteningagent, methyl and propyl-parabens as preservatives, a dye and flavoring.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic. Additionally, thecompounds of Formula I or Formula II, and a salt thereof, can beincorporated into sustained-release preparations and formulations.

The compounds of Formula I or Formula II, or a salt, or a hydratethereof, can be administered to the central nervous system, parenterallyor intraperitoneally. Solutions of the compound as a free base or apharmaceutically acceptable salt can be prepared in water mixed with asuitable surfactant, such as hydroxypropylcellulose. Dispersions alsocan be prepared in glycerol, liquid polyethylene glycols and mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations can contain a preservative and/or antioxidants toprevent the growth of microorganisms or chemical degeneration.

The pharmaceutical forms suitable for injectable use include, withoutlimitation, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Compounds of the present invention may be contained within, mixed with,or associated with, a suitable (acceptable) pharmaceutical carrier foradministration to a patient according to the particular route ofadministration desired. Suitable or acceptable pharmaceutical carriersrefer to any pharmaceutical carrier that will solubilize the compoundsof the present invention and that will not give rise to incompatabilityproblems, and includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, absorptiondelaying agents, and the like. The use of such suitable or acceptablepharmaceutical carriers is well known by those skilled in the art.Preferred carriers include sterile water, physiologic saline, and fivepercent dextrose in water. Examples of other suitable or acceptablepharmaceutical carriers include, but are not limited to, ethanol, polyol(such as propylene glycol and liquid polyethylene glycol), suitablemixtures thereof, or vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size (in the case of adispersion) and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the compoundof Formula I or Formula II in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the sterilized compound of Formula I orFormula. II of this invention into a sterile vehicle that contains thebasic dispersion medium and any of the other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze drying.

Pharmaceutical compositions which are suitable for administration to thenose and buccal cavity include, without limitation, self-propelling andspray formulations, such as aerosol, atomizers and nebulizers.

The therapeutic compounds of Formula I and Formula II, or a salt, or ahydrate thereof, can be administered to a patient alone or incombination with pharmaceutically acceptable carriers or aspharmaceutically acceptable salts, the proportion of which is determinedby the solubility and chemical nature of the compound, chosen route ofadministration to the patient and standard pharmaceutical practice.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided, comprising administering a therapeuticallyeffective amount of a compound of Formula I, or a salt of the compoundof Formula I, or a hydrate of a compound of Formula I, to the patient:

wherein X is selected from the group consisting of:

wherein R is an alkyl group having from one to ten carbon atoms, andwherein R₂ is a halogen atom. In a preferred embodiment of thisinvention, the method of treating a patient having cancer includesadministering a therapeutically effective amount of the compound ofFormula I to the patient wherein the compound of Formula I includeswherein R is a methyl group and R₂ is a halogen atom. In a morepreferred embodiment of this invention, the method of treating a patienthaving cancer includes administering to the patient a therapeuticallyeffective amount of the compound of Formula I to the patient wherein Ris an alkyl group having from one to ten carbon atoms and R₂ is saidhalogen atom that is a chlorine. In a most preferred embodiment of thisinvention, the method of treating a patient having cancer includesadministering to the patient a therapeutically effective amount of thecompound of Formula I to the patient wherein R is a methyl group and R₂is said halogen atom that is a chlorine.

FIG. 2 shows compounds 1, 2, and 3 that are labeled lead compounds thatwere previously set forth in 2012, by Gangjee et al.^(13a,13b, and 13c, respectively.) Gangjee et al., 2012, reportedpyrrolo[3,2-d]pyrimidine known Compound 1 (FIG. 2) that inhibited thebinding of radiolabeled colchicine to the tubulin. Known Compound 1 alsoinhibits the growth of tumor cells with GI₅₀ values in the nanomolarrange and also circumvents Pgp and βIII-tubulin mediated resistancemechanisms that limit the activity of several microtubule targetingagents.^(13a) Known Compound 2 (FIG. 2) with a Cl at the 2-position, wasfound to be much more active than known Compound 1 (Table 1). The5-methyl analog known Compound 3 was slightly more active than knownCompound 2, indicating that a small group is preferred at the5-position.^(13b) In the present invention, compound5-methyl-pyrazolo[4,3-d]pyrimidines, compounds of this invention, andthose identified herein as compounds 4-11 are presented in FIG. 2.Compounds 4-11 of this invention have a Cl (chlorine) group at the2-position of pyrazolo[4,3-d]pyrimidine. The present invention exploresthe SAR (structure activity relationship) of substitution in the anilinoring. While not bound to any particular theory, molecular modelingsuggests that, the additional N2-nitrogen at pyrazolo[4,3-d]pyrimidineacts as a hydrogen bond donor (HBD) which is expected to improve bindingwith the colchicine site. Superimposition of the docked poses of knownCompound 3 in the colchicine site of tubulin at the interface of theα-subunit and ß-subunit of tubulin; PDB: 4O2B¹⁴ using Maestro 2018¹⁵,were carried out (Docking score=−8.69 Kcal/mol). Superimposition of thedocked poses of Compound 4 of this invention in the colchicine site oftubulin at the interface of the α-subunit and ß-subunit of tubulin; PDB:4O2B¹⁴ using Maestro 2018¹⁵, were carried out (Docking score=−9.52Kcal/mol). The N2-position of pyrazolo[4,3-d]pyrimidine is located 4.40A° away from Ala317 backbone carbonyl and will be interacted with theenzyme by hydrogen bonding. This specific interaction is absent in knownCompound 3.

In the following paragraphs, compounds 4-11 of Part I: Synthesis SchemeI, and compounds 6a-6d and compounds 6d1-6d9 of Part II: SynthesisScheme, are described as non-limiting examples, of the compounds of thepresent invention, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

Part I: Synthesis Scheme I

PART I: Synthesis Scheme I, shows that 4-nitro-1H-pyrazole-3-carboxylicacid (compound 12) was alkylated with methyl iodide in the presence ofpotassium carbonate in DMF. After flash chromatography on silica gel,two regioisomers, alkyl 1-alkyl-4-nitro-1H-pyrazole-3-carboxylate(compound 13a, 1,3-isomer) and alkyl1-alkyl-4-nitro-1H-pyrazole-5-carboxylate (compound 13b, 1,5-isomer)were obtained in 26% and 74% yield, respectively. The structures of theisomers (compounds 13a, 13b) were determined by NOESY analysis: a NOESYcorrelation was observed between H-5 and alkyl protons in compound13a.¹⁶ The nitro group of compounds 13a and 13b was reduced byhydrogenation in the presence of a catalytic amount of catalytic amountof 10% Pd/C to produce amines compounds 14a and 14b, respectively.Cyclization of compound 14b with urea afforded compound 15. Chlorinationof compound 15 with POCl₃ and DIPEA in toluene afforded compound 16 in68% yield. Treatment with appropriate substituted anilines inisopropanol with 1 drop of conc. HCl with compound 16, provided theinstant compounds 4-11 of this invention (65-78% yields).

TABLE 1 Biological activity Compound activity in a P-gp Inhibition ofcolchicine binding overexpressing cell line^(a) 5 μM 0.5 μM Inhibitionof tubulin OVCAR-8 NCl/ADR-RES inhibitor % inhibitor % assembly IC₅₀Compounds IC₅₀ (nM) ± SD IC₅₀ (nM) ± SD inhibition ± SD inhibition ± SD(μM ± SD) Paclitaxel 7.0 ± 2  >5,000 — — — CS-A4  3.5 ± 0.7 2.3 ± 0.4 98± 0.1 80 ± 0.6 0.54 ± 0.06 1 (RP/AG/159-124) 1000 ± 300 700 ± 200 95 ±0.8 88 ± 0.7  10 ± 0.6 2 (KS/AG/174-277)  28 ± 4.0  15 ± 4.5 96 ± 0.5 80± 3  2.8 ± 0.7 3 (KS/AG/174-366)  2.5 ± 0.7 1.3 ± 0.4 95 ± 0.4 97 ± 2.0 1.1 ± 0.03 4 (FI/AG/177-359)  9.0 ± 0.7 5.0 ± 0  94 ± 3  68 ± 3  9.0 ±0.7 5 (FI/AG/177-367) 31 ± 2 17 ± 3  93 ± 0.5 64 ± 2  0.82 ± 0.02 6(FI/AG/177-353) 17 ± 2 8.0 ± 0.7 94 ± 0.4 68 ± 2  0.42 ± 0.07 7(FI/AG/177-456) 14 ± 3  11 ± 0.7 91 ± 1  66 ± 0.7 0.49 ± 0.01 8(FI/AG/177-457) 2.0 ± 1  4.0 ± 1  99 ± 0.4 88 ± 0.8 0.42 ± 0.07 9(FI/AG/177-458) 49.0 ± 10   44 ± 8.0 85 ± 1  —  1.1 ± 0.08 10(FI/AG/177-463) 880 ± 40 730 ± 100 64 ± 5  — 2.9 ± 0.2 11(FI/AG/177-455) >5000  >5000 7.1 ± 3  — >20

Compounds 4-11 of this invention were designed and tested to study thedirect effects on tubulin assembly and inhibition of colchicine binding(see Table 1). Except for compounds 9-11, all the compounds at 5 μMinhibited [³H] colchicine binding to the protein, and the extent ofinhibition of all the compounds were similar to that obtained with CA-4.Known Compound 2 inhibited tubulin assembly with 3-fold improvedactivity comparable to the lead known Compound 1. Molecular modelingsuggested that (not shown here) 2-Cl of known Compound 2, interactedwith the hydrophobic pocket created by Leu248 and Ala 246 amino acids.The 5-Me of lead known Compound 3 is oriented towards the hydrophobicpocket of Ala316, Ala317 and Val315 (not shown). These hydrophobicinteractions clearly explained the improved tubulin assembly whencomparing known Compound 3 with known Compound 2. Compounds 4-11 of thisinvention were designed based on the bioisosteric replacement of2-chloro-N,5-dimethyl-1-pyrrolo[3,2-d]pyrimidine scaffold to5-chloro-1-methyl-pyrazolo[4,3-d]pyrimidine scaffold. Compounds 4-11 ofthe present invention inhibited the binding of [³H] colchicine totubulin by 64-99%, whereas the lead known Compound 1 showed 95%inhibition of [3H]colchicine binding.

Compound 4 of the present invention unexpectedly showed (exhibited) a2-fold improvement in inhibition of tubulin assembly over known Compound3. While not bound to any particular theory, the additional N2-nitrogenat pyrazolo[4,3-d]pyrimidine acts as a hydrogen bond donor (HBD) whichmay explain the unexpected improvement in inhibition of tubulin assemblyand binding with the colchicine site. Compound 11 of the presentinvention, which is the NH-hydrogen on N-4 position of known compound 4was not active in OVCAR-8 and NCI/ADR-RES cell lines and did not showcolchicine inhibition. In compound 11 the sigma bonds (C_(1′)—N andN—C₄) connecting the phenyl ring and pyrazolo[4,3-d]pyrimidine ring areboth freely rotatable, while these bonds are somewhat restricted inknown compound 4 where an additional methyl group was introduced on theN-4 position. The ¹H NMR spectrum of known compound 4 (not set forthherein) shows the 5-CH₃ proton in known compound 4 (δ 3.79 ppm) is moreshielded than in compound 11 (δ 4.35 ppm, ¹H NMR spectrum not set forthhere). Due to the bulk of the 4-N-methyl group, the conformations ofknown compound 4 is restricted such that the phenyl ring isconformationally positioned on top of the 5-CH₃ (compound 4). This ¹HNMRstudy defines the aniline ring orientation, in the N4-CH₃ analogs ofcompound 4, to be oriented on the side of the pyrazolo ring of thepyrazolo[4,3-d]pyrimidine scaffold.

Compound 5 of this invention, where the 4′-methoxy aniline of knowncompound 4 was substituted with 4′-S-methylaniline resulted in reducedinhibition in tubulin assembly and colchicine binding. This dataindicates that bioisosteric replacement of electronegative oxygen of the4′-OMe group with sulfur in compound 5 is detrimental to tubulinassembly and colchicine binding. Restriction of the N-4 methyl ofcompound 4 of this invention as a 6-methoxy tetrahydroquinoline ring ascompound 6 of this invention and with a 5′-methoxy-2′-N-methyl naphthylmoiety as compound 7 of this invention have parallel inhibition intubulin assembly and colchicine binding.

Homologation of 4′-OMe (known compound 4) to 4′-OEt (compound 8 of thisinvention) unexpectedly improves OVCAR-8 cell line activity around4-fold over known compound 4 and comparable activity of tubulin assemblyand colchicine binding inhibition with known compound 4. When bulky sidechains —OCH(CH₃)₂ and —OCF₃ (compounds 9 and 10, of this invention,respectively) were introduced at 4′-position OVCAR-8 and NCI/ADR-REScell line activities were diminished. Tubulin assembly and colchicineinhibition were also decreased. Molecular Modeling revealed that due tothe steric clashes of the bulky side chains with Met259 in thecolchicine site of the protein (PDB: 402b, figure not shown) theactivities are decreased.

Part II: Synthesis Scheme I

N-4 substituted Pyrazolo[4,3-d]pyrimidine compounds as MicrotubuleTargeting Agents:

Part II: Synthesis Scheme I

General synthesis for substituted-N-methylanilines: According toTeichert et al.¹⁷ substituted aniline (5-10 mmol) NaOMe (5 equiv) wasadded to a suspension of in MeOH (8-15 mL). The resulting solution waspoured into a suspension of paraformaldehyde (1.4 equiv) in MeOH (5-10mL). The reaction mixture was stirred for 5 hours at RT and then sodiumborohydride (1 equiv) was added. The solution was heated to reflux for 2h. After evaporating part of the solvent, the reaction mixture wastreated with 1 M KOH. The product was extracted with diethyl ether,dried (anhydrous Na₂SO₄). Silica gel (3-5 g) was added followed byevaporation of the solvent under reduced pressure to afford a plug whichwas loaded on a silica column and eluted with Hexanes/EtOAc 2:1. Thefractions containing the product spot (TLC) were pooled and evaporatedunder reduced pressure to afford substituted-N-methylanilines. Generalsynthesis for alkyl 1-alkyl-4-nitro-1H-pyrazole-5-carboxylate (2a) andalkyll 1-alkyl-4-nitro-1H-pyrazole-3-carboxylate (2b):4-nitro-1H-pyrazole-3-carboxylic acid ester (1) was alkylated withexcess alkyl iodide in the presence of potassium carbonate in DMF. Afterflash chromatography on silica gel, two regioisomers, alkyl1-alkyl-4-nitro-1H-pyrazole-3-carboxylate (2a, 1,3-isomer) and alkyl1-alkyl-4-nitro-1H-pyrazole-5-carboxylate (2b, 1,5-isomer) were obtainedin 26% and 74% yield, respectively. The structures of the isomers (2a,2b) were determined by NOESY analysis: a NOESY correlation was observedbetween H-5 and alkyl protons in 2a. The nitro group of 2a and 2b wasreduced by hydrogenation in the presence of a catalytic amount of 10%Pd/C to produce amines 3a and 3b, respectively. General procedure forsynthesis of 4-6: The synthesis of target compounds 4 (Part II: SchemeI), commenced from cyclization of 3b with HCl (g) and acetonitrile,formamide, chloro-formamidine hydrochloride and urea to afford 4a-4d,respectively. Chlorination of 4a-4d with POCl₃ and TEA in tolueneafforded 5a-5d in 68-75% yield. Treatment with appropriate substitutedanilines in isopropanol with 1 drop of conc. HCl provided compounds6a-6d of this invention (65-78% yields).

Synthesized Compounds: (6d1-6d9): See Table 2 for Complete ChemicalStructure

5,7-dichloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidine (5d): ¹H NMR (400MHz, DMSO-d₆) δ 7.48 (s, 1H), 3.95 (s, 3H)

5-chloro-N-(4-methoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d1): ¹H NMR (400 MHz, DMSO-d₆) δ 8.05 (s, 1H), 7.25 (d, J=8.9 Hz, 2H),7.00 (d, J=8.9 Hz, 2H), 3.79 (s, 3H), 3.49 (s, 3H), 3.04 (s, 3H).

5-chloro-N,1-dimethyl-N-(4-(methylthio)phenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d2): ¹H NMR (400 MHz, DMSO-d₆) δ 8.09 (s, 1H), 7.31 (d, J=8.5 Hz, 2H),7.24 (d, J=11.7 Hz, 2H), 3.35 (s, 3H), 3.09 (s, 3H), 2.49 (s, 3H).

1-(5-chloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-6-methoxy-1,2,3,4-tetrahydroquinoline(6d3): ¹H NMR (400 MHz, DMSO-d₆) δ 8.12 (s, 1H), 6.93 (s, 1H), 6.74 (d,J=10.7 Hz, 2H), 4.08-3.82 (m, 2H), 3.75 (s, 3H), 3.21 (s, 3H), 2.91-2.74(m, 1H), 2.17-1.94 (m, 2H).

5-chloro-N-(5-methoxynaphthalen-2-yl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d4) ¹H NMR (500 MHz, DMSO-d₆) δ 8.20 (d, J=9.0 Hz, 1H), 8.10 (s, 1H),7.68 (d, J=2.3 Hz, 1H), 7.51 (dd, J=9.0, 2.3 Hz, 1H), 7.43 (t, J=7.9 Hz,1H), 7.40-7.27 (m, 1H), 6.98 (dd, J=7.8, 0.9 Hz, 1H), 3.97 (s, 3H), 3.63(s, 3H), 2.92 (s, 3H).

5-chloro-N-(4-ethoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d5) ¹H NMR (500 MHz, DMSO-d₆) δ 8.04 (s, 1H), 7.27-7.16 (m, 2H),7.05-6.92 (m, 2H), 4.04 (q, J=7.0 Hz, 2H), 3.48 (s, 3H), 3.03 (s, 3H),1.32 (t, J=6.9 Hz, 3H).

5-chloro-N-(4-isopropoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d6) ¹H NMR (500 MHz, DMSO-d₆) δ 8.05 (s, 1H), 7.21 (d, J=10.2 Hz, 2H),6.98 (d, J=10.2 Hz, 2H), 4.76-4.51 (m, 1H), 3.48 (s, 3H), 3.05 (s, 3H),1.27 (d, J=7.4 Hz, 6H).

5-chloro-N-(3-fluoro-4-methoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d7) ¹H NMR (400 MHz, DMSO-d₆) δ 6.94 (s, 1H), 6.48-6.32 (m, 1H),6.26-6.16 (m, 1H), 6.02 (s, 1H), 3.70 (s, 3H), 3.38 (s, 3H), 2.61 (s,3H).

5-chloro-N,1-dimethyl-N-(4-(trifluoromethoxy)phenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d8) ¹H NMR (500 MHz, DMSO-d₆) δ 7.98 (s, 1H), 7.47 (d, J=9.0 Hz, 2H),6.98 (d, J=9.0 Hz, 2H), 4.31 (s, 3H), 3.80 (s, 3H).

5-chloro-N-(4-methoxyphenyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine(6d9) ¹H NMR (500 MHz, DMSO-d₆) δ 7.59 (s, 1H), 7.28 (d, J=9.0 Hz, 2H),6.82 (d, J=9.0 Hz, 2H), 3.95 (s, 3H), 3.31 (s, 3H).

TABLE 2 data MIA PaCa-2 cell lines and inhibition of colchicine bindingInhibition of colchicine Compound activity in a P-gp bindingoverexpressing cell line^(a) 5 μM NCI/ADR- inhibitor 0.5 μm Inhibitionof OVCAR-8 RES % inhibitor % tubulin IC₅₀ (nM) ± IC₅₀ (nM) ± inhibition± inhibition ± assembly IC₅₀ MIA PaCa-2 Compounds SD SD SD SD (μM ± SD)(nM ± SD) Paclitaxel 7.0 ± 2   >5,000 — — — — CS-A4 3.5 ± 0.7 2.3 ± 0.4 98 ± 0.1  80 ± 0.6 0.54 ± 0.06 —

  6d1 9.0 ± 0.7 5.0 ± 0   94 ± 3  68 ± 3  0.45 ± 0.09 22.67 ± 1.128

  6d2 31 ± 2  17 ± 3   93 ± 0.5 64 ± 2  0.82 ± 0.02 20.92 ± 0.651

  6d3 17 ± 2  8.0 ± 0.7  94 ± 0.4 68 ± 2  0.42 ± 0.07 47.97 ± 2.775

  6d4 14 ± 3   11 ± 0.7 91 ± 1   66 ± 0.7 0.49 ± 0.01

  6d5 2.0 ± 1   4.0 ± 1    99 ± 0.4   88 ± 0.007 0.42 ± 0.07

  6d6 49.0 ± 10    44 ± 8.0 85 ± 1   1.1 ± 0.08

  6d7 9.0 ± 1   7.0 ± 3    97 ± 0.3 77 ± 1  0.74 ± 0.04

  6d8 880 ± 40  730 ± 100 64 ± 5  2.9 ± 0.2

  6d9 200 ± 40  150 ± 0  74 ± 2  1.2 ± 0.2 GEM — — — — — 296.8 ± 60.4 PMX — — — — — 16.1 ± 1.4 

TABLE 3 MCF-7-TUBB3 RESISTANCE RESISTANCE MCF-7 WT β3-tubulin RATIOMCF-7/TAX RATIO Agent EC₅₀ (nM) EC₅₀ (nM) β3-tubulin/WT Pgp EC₅₀ (nM)Pgp/WT 6d1 2.0 ± 0.3 3.3 ± 0.4 1.1 15.1 ± 2.0 3.8 6d4 35.2 ± 4.7  16.8 ±1.5  0.5 43.0 ± 7.2 1.2 6d5 7.7 ± 0.7 9.7 ± 1.0 1.3 31.3 ± 3.5 4.0 6d71.7 ± 0.2 2.6 ± 0.4 1.6  8.1 ± 0.2 4.8 PACLITAXEL 194.2 2065.8 10.63225.0 16.6

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It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A compound of Formula II:

wherein R₁ is selected from the group consisting of a hydrogen, an alkylgroup having from one to six carbon atoms, an aryl group, and aheteroaryl group; R₂ is selected from the group consisting of an alkylgroup having from one to six carbon atoms, a hydrogen, a NR^(a)R^(b)group wherein R^(a) is either a hydrogen or an alkyl group having fromone to six carbon atoms and R^(b) is either a hydrogen or an alkyl grouphaving from one to six carbon atoms, an aryl group, a heteroaryl group,and a halogen; R is an alkyl group having from one to six carbon atoms;and Ar is selected from the group consisting of an alkyl group havingfrom one to six carbon atoms, a substituted aryl, and a substitutedheteroaryl.
 2. The compound of claim 1 wherein R₂ is either a methylgroup, NH₂, or a chlorine.
 3. The compound of claim 2 that is oneselected from the group consisting of5-chloro-N-(4-methoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N,1-dimethyl-N-(4-(methylthio)phenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;1-(5-chloro-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-yl)-6-methoxy-1,2,3,4-tetrahydroquinoline;5-chloro-N-(5-methoxynaphthalen-2-yl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N-(4-ethoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N-(4-isopropoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N-(3-fluoro-4-methoxyphenyl)-N,1-dimethyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine;5-chloro-N,1-dimethyl-N-(4-(trifluoromethoxy)phenyl)-1H-pyrazolo[4,3-d]pyrimidin-7-amine;and5-chloro-N-(4-methoxyphenyl)-1-methyl-1H-pyrazolo[4,3-d]pyrimidin-7-amine.4. A pharmaceutical composition comprising a compound of Formula II

wherein R₁ is selected from the group consisting of a hydrogen, an alkylgroup having from one to six carbon atoms, an aryl group, and aheteroaryl group; R₂ is selected from the group consisting of an alkylgroup having from one to six carbon atoms, a hydrogen, a NR^(a)R^(b)group wherein R^(a) is either a hydrogen or an alkyl group having fromone to six carbon atoms and R^(b) is either a hydrogen or an alkyl grouphaving from one to six carbon atoms, an aryl group, a heteroaryl group,and a halogen; R is an alkyl group having from one to six carbon atoms;and Ar is selected from the group consisting of an alkyl group havingfrom one to six carbon atoms, a substituted aryl, and a substitutedheteroaryl.
 5. The pharmaceutical composition of claim 4 including apharmaceutically acceptable carrier.
 6. A method of treating a patienthaving cancer comprising administering to said patient a therapeuticallyeffective amount of a compound of Formula II, or optionally a salt or ahydrate of said compound of Formula II:

wherein R₁ is selected from the group consisting of a hydrogen, an alkylgroup having from one to six carbon atoms, an aryl group, and aheteroaryl group; R₂ is selected from the group consisting of an alkylgroup having from one to six carbon atoms, a hydrogen, a NR^(a)R^(b)group wherein R^(a) is either a hydrogen or an alkyl group having fromone to six carbon atoms and R^(b) is either a hydrogen or an alkyl grouphaving from one to six carbon atoms, an aryl group, a heteroaryl group,and a halogen; R is an alkyl group having from one to six carbon atoms;and Ar is selected from the group consisting of an alkyl group havingfrom one to six carbon atoms, a substituted aryl, and a substitutedheteroaryl.
 7. A method of treating a patient having cancer comprisingadministering to said patient a therapeutically effective amount of apharmaceutical composition comprising a compound of Formula II, oroptionally a salt or a hydrate of said compound of Formula II

wherein R₁ is selected from the group consisting of a hydrogen, an alkylgroup having from one to six carbon atoms, an aryl group, and aheteroaryl group; R₂ is selected from the group consisting of an alkylgroup having from one to six carbon atoms, a hydrogen, a NR^(a)R^(b)group wherein R^(a) is either a hydrogen or an alkyl group having fromone to six carbon atoms and R^(b) is either a hydrogen or an alkyl grouphaving from one to six carbon atoms, an aryl group, a heteroaryl group,and a halogen; R is an alkyl group having from one to six carbon atoms;and Ar is selected from the group consisting of an alkyl group havingfrom one to six carbon atoms, a substituted aryl, and a substitutedheteroaryl.
 8. The method of claim 7 including wherein saidpharmaceutical composition includes a pharmaceutically acceptablecarrier.